award number: w81xwh-04-1-0500 principal …modified porphyrins as photosensitizers for the...

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Award Number: W81XWH-04-1-0500 TITLE: Structure Optimization of 21,23-Core-Modified Porphyrins Absorbing Long-Wavelength Light as Potential Photosensitizers Against Breast Cancer Cells PRINCIPAL INVESTIGATOR: Michael R. Detty, Ph.D. CONTRACTING ORGANIZATION: New York State University Amherst, NY 14228-2567 REPORT DATE: April 2007 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.

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3. DATES COVERED 1 Apr 2006 – 31 Mar 2007

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Structure Optimization of 21,23-Core-Modified Porphyrins Absorbing Long-Wavelength Light as Potential Photosensitizers Against Breast Cancer Cells

5b. GRANT NUMBER W81XWH-04-1-0500

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6. AUTHOR(S)

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Michael R. Detty, Ph.D.

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Email: [email protected]

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New York State University Amherst, NY 14228-2567

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14. ABSTRACT In the first year, we made eighteen new 21,23-core modified porphyrins, determined their photophysical properties, and evaluated the biological properties of them. In the second year, the research focused on 1) analyzing quantitative structure-activity relationships (QSAR) establishing new synthetic methods to prepare eleven new second generation derivatives with novel structures. During the past (third) year, we have evaluated the new derivatives structurally, photophysically, and biologically. The structures of two derivatives were determined unambiguously be x-ray crystallography including the structure of a cis-ABCC meso-substituted derivative and the structure of a cis-AB disubstituted derivative. A series of carboxylic acid-substituted dithiaporphyrins was prepared with different length aliphatic spacers between porphyrin and acid. In a collaborative study with Prof. Benny Ehrenberg at Bar Ilan University in Israel, we found that the efficiency of photooxidizing a membrane-residing singlet oxygen target decreases as the side chains become longer. This has implications for fine-tuning optimal structures for PDT sensitizers. The most promising dithiaporphyrin from in vitro studies is being evaluated in toxicity studies in vivo.

15. SUBJECT TERMS Photodynamic therapy, breast cancer,core-modified porphyrin, in vitro biological activity

16. SECURITY CLASSIFICATION OF:

17. LIMITATION OF ABSTRACT

18. NUMBER OF PAGES

19a. NAME OF RESPONSIBLE PERSON USAMRMC

a. REPORT U

b. ABSTRACT U

c. THIS PAGE U

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19b. TELEPHONE NUMBER (include area code)

Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18

Table of Contents

Cover……………………………………………………………………………………1 SF 298……………………………………………………………………………..……2 Table of Contents………….…………………………………………………..…..…3

Introduction…………………………………………………………….…………....4

Body…………………………………………………………………………………….4 Key Research Accomplishments………………………………………….………4 Reportable Outcomes……………………………………………………………….5 Conclusions…………………………………………………………………….……..5 References…………………………………………………………………………..…5 Appendices……………………………………………………………………….…...5

Introduction The objectives of this project are two fold: one was to train the former PI, Dr. Youngjae You, as an photodynamic cancer therapy expert in breast cancer research and the other is to perform the research to optimize the structure of 21,23-core-modified porphyrins as potential photosensitizers that are able to absorb long-wavelength light for treating breast cancer. Dr. You has successfully completed his postdoctoral studies and is now a tenure-track assistant professor at the University of South Dakota. His research interests at the University of South Dakota are focused on the synthesis and study of core-modified porphyrins as photosensitizers for the photodynamic therapy of cancer. In the first year, we made eighteen new 21,23-core modified porphyrins, determined their photophysical properties, and evaluated the biological properties of them. In the second year, the research focused on 1) analyzing quantitative structure-activity relationships (QSAR) establishing new synthetic methods to prepare eleven new second generation derivatives with novel structures. In the second year, the research focused on 1) analyzing quantitative structure-activity relationships (QSAR) establishing new synthetic methods to prepare eleven new second generation derivatives with novel structures. Body Work initiated in the past year is ongoing and will be completed during the additional time afforded by the no cost extension. A series of carboxylic acid-substituted dithiaporphyrins was prepared with different length aliphatic spacers between porphyrin and acid. These 4-(carboxyalkyl)phenyl substituted derivatives had spacers of 1-10 methylene (CH2) units. These were prepared via chemistry that was developed in the first two years of this award. In a collaborative study with Prof. Benny Ehrenberg at Bar Ilan University in Israel, we found that the efficiency of photooxidizing a membrane-residing singlet oxygen target decreases as the side chains become longer. We suspect that with these large molecules, when their carboxylates are anchored at the lipid:water interface, the tetrapyrrole is in fact closer to the other, opposite, side of the bilayer. This make these molecules interesting and different from the all the others that have been examined in the literature. Conclusions regarding the location of these materials in the bilayer await the synthesis/purchase of phospholipids that are tagged with fluorophores at their heads. The structures of two derivatives were determined unambiguously be x-ray crystallography including the structure of a cis-ABCC meso-substituted derivative and the structure of a cis-AB disubstituted derivative. Details of these crystal structures are contained in the appendix in the J. Porphyrins Phthalocyanines article. The characterization of the cis-ABCC derivative will allow this superior substituent pattern to be isolated unequivocally. The role of the dithiaporphyrins in inducing apoptosis was determined. This work is summarized in the appendix in the J. Photochem. Photobiol. B article. The success of the photosensitizer, I-YY-69, in vitro prompted the design of in vivo studies to determine 1) its dark toxicity and 2) its efficacy in photodynamic therapy if acceptable toxicity is observed. These studies are ongoing. Key Research Accomplishments Training: Dr. You has successfully completed his postdoctoral studies and is now a tenure-track assistant professor at the University of South Dakota. His research interests at the University of South Dakota are focused on the synthesis and study of core-modified porphyrins as photosensitizers for the photodynamic therapy of cancer. Research accomplishments: 1) Synthesis of diverse sets of core-modified porphyrins, 2) determination of physical properties of prepared porphyrins, 3) evaluation of biological effects of the compounds and analysis of structure-activity relationships, 4) elucidating of apoptotic cell death after PDT treatment, and 5) unambiguously determining the structure of two new classes of dithiaporphyrins using x-ray crystallography, and 6) preparing a series of substituted derivatives to probe depth of penetration of the core-modified porphyrins in the lipid bilayer..

Reportable Outcomes Publications: Part of this report was published in two separate publications included in the appendix at the end of the report; a) You, Y.; Daniels, T. S.; Dominiak, P. M.; Detty, M. R. “Synthesis, Spectral Data, and Crystal Structure of Two Novel Substitution Patterns in Dithiaporphyrins,” J. Porphyrins Phthalocyanines 2007, 11, 1-8. b) You, Y.; Gibson, S. L.; Detty, M. R. “Phototoxicity of a Core-modified Porphyrin and Induction of Apoptosis,” J. Photochem. Photobiol. B 2006, 85, 155-162. Poster: Part of Dr. You’s work was presented in one poster: Gannon, Michael K.; Tombline, Gregory; Donnelly, David J.; Holt, Jason J.; You, Youngjae; Ye, Mao; Nygren, Cara L.; Detty, Michael R. “Characterization of the “R” Binding Site of P-glycoprotein: Using Novel Chalcogenoxanthylium Tetramethylrosamine Analogs for the Stimulation of ATPase Activity,” 232nd National Meeting of the American Chemical Society, San Francisco, CA (2006). Conclusions Dr. You’s postdoctoral studies were completed successfully and he is now employed as an assistant professor in the Department of Chemistry at South Dakota State University. Biological and synthetic studies will be concluded during the coming year with work at both the University at Buffalo and at South Dakota State University. References N/A Appendices J. Porphyrins Phthalocyanines 2007, 11, 1-8. J. Photochem. Photobiol. B 2006, 85, 155-162.

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M

Copyright © 2007 Society of Porphyrins & Phthalocyanines

Synthesis, spectral data, and crystal structure of two novel substitution patterns in dithiaporphyrins

Youngjae You*a, Thalia S. Danielsb, Paulina M. Dominiakb and Michael R. Dettyb∏

a Department of Chemistry & Biochemistry, South Dakota State University, Brookings, South Dakota 57007-0896, USA b Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000, USA

Received 1 September 2006Accepted 2 November 2006

ABSTRACT: The syntheses, characterizations and crystal structures of 5-(4-tert-butylphenyl)-15,20-diphenyl-10-(4-methoxy)phenyl-21,23-dithiaporphyrin (1b) and 2,3-dimethoxy-10,15-diphe-nyl-21,23-dithiaporphyrin (2) are described. The cyclization of 2-[1-(4-methoxyphenyl)-1-pyrrolo-methyl]-5-(1-phenyl-1-pyrrolomethyl)thiophene and 2-[1-(4-t-butylphenyl)-1-hydroxymethyl]-5-(1-phenyl-1-hydroxymethyl)thiophene gave predominantly the cis-regioisomer, which was characte-rized by X-ray crystallography. Copyright © 2007 Society of Porphyrins & Phthalocyanines.

KEYWORDS: core-modified porphyrin, synthesis, X-ray crystal structure, spectral data.

INTRODUCTIONPorphyrin derivatives have been developed or

are under development as photosensitizers for photo-dynamic therapy due to the favorable photophysical characteristics of porphyrins: absorbing light of 630 nm and generating singlet oxygen effectively [1, 2]. However, photosensitizers in deeper tissues can only be activated by light that penetrates, which is preferably in the 650-800 nm range [3]. The subs-titution of the core nitrogen atom(s) of natural porphyrins with heavy atoms such as S, Se, and Te provides a red-shift in their absorption spectra [4-7]. The 21,23-dithiaporphyrin core, especially, is an excellent pharmacophore for good photosensitizers, absorbing light of about 700 nm as well as genera-ting singlet oxygen effectively [5]. Some dithiapor-phyrins having two carboxylic acids expressed sub-micromolar phototoxicity in vitro when activated with minimal light 5 J.cm-2 of filtered, 350-750 nm broad band light [8-10].

While the core atoms of porphyrins affect their photophysical properties, the structure and placement of meso substituents might influence the binding with biological components such as membranes, enzymes, receptors, and lipoproteins. The binding with possible molecular targets might subsequently determine the biological efficiency of the porphyrins as photosensitizers. In our studies of structure-acti-vity relationships between core-modified porphyrins and phototoxicity, we clearly observed the effects of the size and the symmetry of the placement of substi-tuents at meso-aryl groups on their phototoxicity [9, 10].

Syntheses of 21,23 core-modified porphyrins were pioneered by Ulman et al. [11-15]. More re-cently, Leeʼs group published a [3 + 1] cyclization method to prepare various types of core-modified porphyrins with [A2B2]-substitution patterns [16, 17] and Chandrashekarʼs group developed a convenient method for the synthesis of expanded core-modified porphyrins [18-20]. However, studies of structure-activity relationships directed toward phototoxicity in core-modified porphyrins are limited by the avai-lable synthetic methods for their preparation. In par-ticular, few synthetic methods are available for the

*Correspondence to: Youngjae You, email: [email protected], fax: +1 605-688-6364

∏SPP full member in good standing

Journal of Porphyrins and Phthalocyanines Published at http://www.u-bourgogne.fr/jpp/

J. Porphyrins Phthalocyanines 2007; 11: 1-8

Published on web 02/06/2007

Copyright © 2007 Society of Porphyrins & Phthalocyanines J. Porphyrins Phthalocyanines 2007; 11: 1-8

Y. YOU ET AL.2

synthesis of dithiaporphyrins with more diverse substituents at the meso-positions. This is especially the case of the 21,23-di-thiaporphyrins of which the synthesis and characterization of cis-[ABC2]- and trans-[ABC2]-substitution patterns in the meso-substituents have not been described [8], and derivatives with unsubstituted meso-substituents are also very scarce [21, 22]. Herein, we report the synthesis, characte-rization, and X-ray crystal structures of two novel dithiaporphyrins: 5-(4-tert-butyl-phenyl)-15,20-diphenyl-10-(4-methoxy)-phenyl-21,23-dithiaporphyrin 1b (a cis-[ABC2] meso-substituent pattern) and 2,3-dimethoxy-10,15-diphenyl-21,23-di-thiaporphyrin 2 (unsubstituted meso substi-tuents).

RESULTS AND DISCUSSION

Synthesis and characterization

The facile [3 + 1] type condensation reaction was adopted in the cyclization for the dithiaporphyrins, 1b and 2. The precursors 4, 5, and 8 were prepared from the aldehydes and thiophene by methods described in our previous study [9]. The yield of the condensation reaction to give compound 7 was poor (27%) relative to the 75% yield for the condensation reaction to give compound 4. From the cyclization reaction of 4 and 5, we expected to get two regioisomeric dithia-porphyrins, trans and cis-forms. Indeed two spots, presumably of two isomers, were observed by thin layer chromatography (TLC). The upper spot was isolated in yields too low to allow characterization.

However, the second product, cis-form (1b), was successfully isolated and charaterized in 18% yield. Spectroscopic data (1H, 13C NMR, and high mass) were consistent with the assigned structure, which was confirmed by X-ray crystal data. On the other hand, the cyclization to give 2 gives a single isomer in 18%. The two meso-hydrogens of dithiaporphyrin 2 gave a characteristic peak in the 1H NMR spectrum at 10.73 ppm while high resolution MS mass data were consistent with the expected molecular formula. The structure of 2 was confirmed by X-ray crystal

data.The UV-vis spectra of compounds 1b,

2, and 9 shown in Fig. 1 are typical core-modified porphyrinic spectra with four weak Q-bands and one strong Soret band in the visible range. For the asymmetric dithia-porphyrin 1b, the properties of the bands, λmaxʼs and εʼs, are very close to those of the symmetric 5,10,15,20-tetraphenyl-21,23-dithiaporphyrin (9) (Table 1) [15]. On the other hand, the electronic absorptions in the spectrum of compound 2 are quite different from those in the spectra of 9 and 1b (Fig. 1). The Soret band is blue-shifted, by about 7 and 9 nm, and its excitation coefficient is smaller, 1.76 × 105 M-1.cm-1, than those of compounds 9 and 1b. While all four Q bands of 2 are blue-shifted, the effects on extinction coefficients are more pronounced

Fig. 1. Electronic absorption spectra of 1b (thin line) and 2 (thick line)

Table 1. Wavelengths and excitation coefficients of electronic absorption spectra of compounds 1b, 2, and 9

S

N N

S

9

9a 1b 2

λmaxb εc λmax ε λmax ε

435 297.5 437 279.3 428 175.5

515 29.6 516 26.6 509 25.6

548 7.3 550 12.4 536 4.0

635 2.2 635 2.0 622 2.4

699 4.6 699 6.0 685 3.8

a 9: 5,10,15,20-tetraphenyl-21,23-dithiaporphyrin, the data of com-pound 9 were adopted from the reference 15; b nm; c 103 M-1.cm-1; the solvents: CH2Cl2 for compounds 1b and 2 and CHCl3 for compound 9.


DITHIAPORPHYRINS 3

in bands I and III, 1.24 and 7.3 × 104 M-1.cm-1 vs 4 × 103 M-1.cm-1 for band III and 6 and 4.6 × 103 M-1.cm-1 vs 3.8 × 103 M-1.cm-1 for band I. These data demonstrate that the number of aryl groups at the meso positions affects the electronic spectra more (9 vs 2) than the molecular symmetry of tetra-aryl dithiaporphyrin (9 vs 1b). In other words, while structural variations between 2 and 9 reside directly on the dithiaporphyrin ring, the structural differences between 1b and 9 are at the para positons of meso aryl rings.

X-ray structures of 1b and 2

The identification of the major isomer isolated from the synthesis shown in Scheme 1 was obtained via X-ray crystallo-graphic analysis. Other spectroscopic me-thods would not be able to assign unequi-vocally the correct regiochemistry to the porphyrin molecule. The black and planar single crystals of 1b were obtained by direct solvent diffusion of n-hexane into a saturated solution in dichloromethane over a 3-week period in a cold room. The dithia-porphyrin core of the molecule is almost planar (0.073 Å of mean deviation from the plane, Fig. 2). The phenyl rings are rotated out of the core plane (55º, 62º, 64º and 79º). The N1…N2 distance is elongated (4.614

Å) compared with a natural four nitrogenic porphyrin (4.042 Å) [23] to accommodate the large S atoms. Two molecules form a dimer in the crystal lattice mainly via C–H...π and π…π interactions with the distance between dithiaporphyrin planes equal to 3.63 Å (Fig. 3). The interplanar distance is slightly longer than the typical separation for aggregated porphyrins which is 3.4-3.6 Å [24]. The centers of the molecules are offset by 5.55 Å which again is outside the usual limits (3-4 Å) for π...π interacting porphyrins. This very weak π...π face-to-face interaction is supported by C–H...π face-to-edge interactions (C(45)–H(31)...N(1) distance equal to 3.345 Å) to form a dimer. Two dichloromethane molecules of solvation, one for each porphyrin 1b, were omitted for clarity in the figure.

The single crystals of compound 2 were also grown by direct solvent diffusion of n-hexane into a saturated solution in di-chloromethane over a week. The black, planar crystals were characterized by X-ray crystallography. The core of the dithia-porphyrin is planar (0.035 Å of mean devia-

Fig. 2. Structure of compound 1b viewed (a) from the top and (b) from the side with hydrogen atoms omitted for clarity. Displace-ment ellipsoids are drawn at the 50% probability level

Fig. 3. The π…π interactions between the two closest molecules in the crystal lattice and the molecular packing diagram of compound 1b. Unit cell axis a shown in red, b in green and c in blue


Y. YOU ET AL.4

tion from the plane, Fig. 4). The phenyl rings and one of the methoxy groups are nearly perpendicular to the core (81º, 64º and 77º, respectively) whereas the second methoxy group is almost in the plane of the core. The N1…N2 distance is also increased (4.584 Å) compared to four nitrogenic porphyrin (4.042 Å) [23]. Two molecules form a dimer in the crystal lattice mainly via π…π interactions (Fig. 5) with the distance between dithiaporphyrin planes equal to 3.41 Å. This intraplanar distance together with 3.55 Å offset is typical for strong cofacial π…π interaction observed in porphyrins [24].

The crystal structures of compounds 1b and 2 show different dimerization patterens. The interac-tion between two molecules of 2 seems to be stronger and the distance between two molecules is shorter, 3.41 Å relative to the dimers of 1b, 3.63 Å. For 2, the dimeric molecules are more overlapped due to reduced steric interactions of the meso-phenyl groups on one molecule with the unsubstituted meso-positions of the other. Interestingly, the dimers of 1b are organized in parallel sheets while the dimers of 2 show perpendicular stacking.

There are three other structures of free base 21,23-dithiaporpyrin derivatives deposited in the Cambrid-ge Structural Database: [25] 5,10,15,20-tetrakis(2-thienyl)-21,23-dithiaporphyrin [26], 21,23-dithiate-traphenylporphyrin [27], and 2,3,12,13-tetrabutoxy-5,10,15,20-tetraphenyl-21,23-dithiaporphyrin [28] (Refcodes: BEHGUR, DTHTPP02 and XUJZUK, respectively). Porphyrin cores overlap only in the

first structure (BEHGUR) with a fairly large interplanar distance (3.71 Å) and off-set (4.83 Å). However, they do not form isolated dimers but rather form a single strand with molecules placed equidistantly from each other. In the remaining structures, distances between the two closest parallel porphyrin cores are much bigger (4.49 and 4.66 Å in DTHTPP02 and XUJZUK, respectively) and the parallel shift is larger than the size of the porphyrine core (10.85 and 7.24 Å for DTHTPP02 and XUJZUK, respectively).

EXPERIMENTAL

Materials

Solvents and reagents were used as received from Sigma-Aldrich Chemical Co. (St. Louis, MO) unless otherwise noted. Concentration in vacuo was performed on a Buchi rotary evaporator. NMR spectra were recorded at 23 °C on a Varian Gemini-300,

Fig. 4. Structure of compound 2 viewed (a) from the top and (b) from the side with hydrogen atoms omitted for clarity. Displacement ellipsoids are drawn at the 50% probability level

Fig. 5. The π…π interactions between the two closest molecules in the crystal lattice and the molecular packing diagram of compound 2. Unit cell axis a shown in red, b in green and c in blue


DITHIAPORPHYRINS 5

Inova 400, or Inova 500 instrument with residual solvent signal as the internal standard: CDCl3 (δ 7.26 for proton, δ 77.16 for carbon) or CD2Cl2 (δ 5.32 for proton, δ 54.00 for carbon). Infrared spectra were recorded on a Perkin-Elmer FT-IR instrument. UV-visible near-IR spectra were recorded on a Perkin-Elmer Lambda 12 spectrophotometer. ESI mass spectrometries were conducted by the Instru-ment Center of the Department of Chemistry at the University at Buffalo.

Synthesis

Compound 6 was prepared as described in the literature [22, 29] and compounds 3, 5, 8 were prepared by methods described in our earlier work [8, 9].

Synthesis of 2-[1-(4-methoxyphenyl)-1-pyrro-lomethyl]-5-(1-phenyl-1-pyrrolomethyl)thiophene 4. Compound 3 (3.64 g, 0.011 mol) was dissolved in excess pyrrole (23 mL, 0.33 mol) and stirred at am-bient temperature for 2 h. Boron trifluoride etherate (0.14 mL, 0.0011 mol) was added and the mixture stirred for another 2.5 h at ambient temperature. The reaction was stopped by the addition of 20 mL of dichloromethane followed by 40% NaOH (15 mL). The organic phase was separated and washed with

distilled water (3 × 15 mL) and brine (1 × 15 mL), dried over MgSO4 and concentrated. Excess pyrrole was removed via vacuum distillation at 80 °C for 2 h. The brown oil was then purified by column chroma-tography on silica gel eluted with a 5:1 mixture of hexanes/ethyl acetate to give 3.20 g (68%) of 4 as a light brown oil. 1H NMR (500 MHz, CDCl3): δH, ppm 3.79 (s, 3 H), 5.52 (s, 1 H), 5.56 (s, 1 H), 5.91 (s, 2 H), 6.14 (s, 2 H), 6.61 (s, 2 H), 6.69 (s, 2 H), 6.84 (d, 2 H, J = 8.8 Hz), 7.15 (d, 2 H, J = 8.4 Hz), 7.23-7.38 (m, 5 H), 7.89 (s, 2H). 13C NMR (75 MHz, CDCl3): δC, ppm 45.7, 46.6, 55.8, 107.9, 108.0, 108.7, 108.8, 114.5, 117.7, 118.2, 125.7, 125.9, 127.6, 128.9, 129.1, 129.9, 133.6, 133.9, 135.4, 143.2, 146.2, 146.9, 159.1. LRMS (ESI): m/z 425.2. Calcd. for C27H24N2OS + H+: 425.2.

Synthesis of 3,4-dimethoxy-2,5-bis[(1-pyrrolo-methyl)]-thiophene 7. Compound 7 was prepared from compound 1 by methods similar to those used for the preparation of compound 4. Yield 27%. 1H NMR (400 MHz, CDCl3): δH, ppm 3.85 (6H, s), 3.93 (4H, s), 5.99 (2H, s), 6.10 (2H, s), 6.67 (2H, s), 8.32 (2H, br s). LRMS (ESI): m/z 303.0. Anal. calcd. for C16H18N2O2S + H+: 303.1.

Synthesis of 5-(4-tert-butylphenyl)-15,20-diphe-nyl-10-(4-methoxy)phenyl-21,23-dithiaporphyrin

Scheme 1. Synthetic schemes for 5-(4-tert-butylphenyl)-15,20-diphenyl-10-(4-methoxy)phenyl-21,23-dithiaporphyrin (1b) and 2,3-dimethoxy-10,15-diphenyl-21,23-dithiaporphyrin (2)

S

N N

S

O

BF3 - etherate

pTsOH, TCBQ

1b

3

pyrrole

5

S

N N

S

O 1a

+

S

NH HN

SOH OH

O

SOH OH

O

4

SO O

HH

OO S

N N

S

HH

O O

S

OO

NH HN6

7

2

SOH OH

8BF3 - etherate

pyrrole pTsOH, TCBQ


Y. YOU ET AL.6

1b. Compound 5 (1.45 g, 0.0041 mol), 4 (1.75 g, 0.0041 mol), and p-toluenesulfonic acid (0.78 g, 0.0041 mol) were dissolved in 510 mL of dichloro-methane. The reaction vessel was covered comple-tely with foil and stirred in the dark at ambient tempe-rature for 0.5 h. Tetrachlorobenzoquinone (TCBQ) (4.05 g, 0.017 mol) was added and the solution was heated at reflux for 1.5 h. The reaction mixture was cooled to ambient temperature, filtered, and concen-trated. The crude product was purified several times via column chromatography on basic alumina eluted with a 1:1 mixture of dichloromethane/hexanes. Porphyrin 1b was isolated as the second red band. The product was washed with minimal acetone several times to retrieve 0.549 g (18%) of 1b as a purple solid. mp >300 °C. 1H NMR (500 MHz, CDCl3): δH, ppm 1.61 (s, 9H), 4.10 (s, 3H), 7.39 (d, 2H, J = 8.5 Hz), 7.82-7.90 (m, 8H), 8.17-8.22 (m, 4H), 8.26 (d, 4H, J = 6.0 Hz), 8.70 (d, 2H, J = 3.2 Hz), 8.73 (d, 2H, J = 3.2 Hz), 9.71 (d, 2H, J = 5.0 Hz), 9.76 (d, 1H, J = 5.0 Hz), 9.77 (d, 1H, J = 5.5 Hz). 13C NMR (75 MHz, CDCl3): δC, ppm 32.2, 35.5, 56.1, 113.6, 125.0, 127.9, 128.5, 134.3, 134.4, 134.5, 134.6, 134.7, 134.7, 135.0, 135.2, 136.0, 136.2, 138.8, 141.9, 148.4, 148.4, 151.5, 156.9, 157.2, 160.3. HRMS (ESI): m/z 735.2693. Anal. calcd. for C49H38ON2

32S2 + H+: 735.2710.Synthesis of 2,3-dimethoxy-10,15-di-

phenyl-21,23-dithiaporphyrin (2). Dithia-porphyrin 2 was prepared from compounds 7 and 8 by methods similar to those used for the preparation of compound 1b. Yield 18%, mp >300 °C. 1H NMR (400 MHz, CD2Cl2): δH, ppm 4.95 (6H, s), 7.88 (6H, s), 8.29 (4H, d, J = 4.0 Hz), 8.82 (2H, d, J = 3.6 Hz), 9.10 (2H, d, J = 3.6 Hz), 9.75 (2H, s), 10.73 (2H, s). 13C NMR (75 MHz, CD2Cl2): δC, ppm 63.2, 113.7, 127.6, 128.3, 134.1, 134.3, 134.5, 135.1, 135.3, 136.6, 141.0, 147.6, 151.0, 155.3, 155.9. HRMS (ESI): m/z 557.1352. Anal. calcd. for C34H24O2N2

32S2 + H+: 557.1352).

X-ray data collection and refinement

X-ray diffraction data on 1b and 2 were collected at 90(1)K using a Brüker SMART APEX2 CCD diffractometer installed at a rotating anode source (MoKα radiation, λ = 0.71073 Å), and equipped with an Oxford Cryosystems nitrogen gas-flow apparatus. The data were collected by the rotation method with 0.5° (0.3° for com-pound 2) frame-width (ω scan) and 35 (20 for compound 2) s exposure time per frame. Four sets of data (360 (600 for compound 2) frames in each set) were

collected, nominally covering complete reciprocal space. The data were integrated, scaled, sorted and averaged using the SMART software package [30]. The structure was solved by Direct methods using SHELXTL NT Version 6.14 [31]. The structure was refined by full-matrix least squares against F2. Non-hydrogen atoms were refined anisotropically. Positions of hydrogen atoms were found by difference electron density Fourier synthesis. The CH3 hydrogens were treated as part of idealized CH3 groups with Uiso = 1.5Ueq, while the remainder of the hydrogen atoms were refined with the “riding” model with Uiso = 1.2Ueq. Structure refinement data are given in Table 2. Atomic coordinates, anisotropic displacement parameters, bond lengths and angles are given in the supporting information section.

CONCLUSIONTwo novel dithiaporphyrins substitution pat-

terns were incorporated in the synthesis and charac-terization of compounds 1b and 2. The structures of 1b and 2 were unambiguously confirmed by X-ray crystallography and the major isomer produced in the preparation of the meso-[ABC2] dithiapor-phyrins 1 was the cis-regioisomer 1b. The first

Table 2. Crystallografic data for 1b and 2

Compound 1b 2

Formula C49H38N2OS2, CH2Cl2 C34H24N2O2S2

Mr, g.mol-1 819.86 556.67

T [K] 90(1) 90(1)

Space group P21/c P21/n

a, Å 13.0893(3) 14.8335(6)

b, Å 15.2550(4) 12.8034(5)

c, Å 20.4068(5) 14.9590(6)

β, ° 95.7150(10) 112.8560(10)

V, Å3 4054.52(17) 2617.94(18)

Z 4 4

ρcalc, g.cm-3 1.343 1.412

μ, mm-1 0.305 0.241

Reflections measured 63002 39987

Unique reflections (Rint) 10082 (0.028) 6490 (0.022)

Parameters refined 531 363

R [I > 2σ(I)] 0.055 0.031

wR2 [I > 2σ(I)] 0.15 0.079

G.O.F. 1.03 1.03


DITHIAPORPHYRINS 7

dithiaporphyrin bearing two unsubstituted meso-positions was also prepared and characterized. The aromatic groups at the meso positions influenced the electronic absorption spectra both in the wavelength and the oscillator strength of the absorption. With these structurally novel dithiaporphyrins in hand, the synthesis of other derivatives will now be facilitated and these substitution patterns will be evaluated for their utility as photosensitizers for photodynamic therapy.

Supporting information

Crystallographic data of compound 1b and 2 including atomic coordinations, equivalent isotropic displacement parameters, anisotropic displace-ment parameters, and bond lengths and angles are available. Crystallographic data have been deposited at the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK and copies can be obtained on request, free of charge, by quoting the publication citation and the deposition numbers 615619 for 1b and 615620 for 2.

Acknowledgements

This research was supported by the Department of Defense [Breast Cancer Research Program] under award number (W81XWH-04-1-0500). Views and opinions of, and endorsements by the author(s) do not reflect those of the US Army or the Department of Defense.

REFERENCES 1. Nyman ES and Hynninen PH. J. Photochem.

Photobiol. B 2004; 73: 1-28. 2. Pandey RK and Zheng G. In The Porphyrin

Handbook Vol. 6, Kadish KM, Smith KM, Guilard R. (Eds.) Academic Press: San Diego, Calif, 2000; pp 157-230.

3. Detty MR. Expert Opin. Ther. Pat. 2001; 11: 1849-1860.

4. Stilts CE, Nelen MI, Hilmey DG, Davies SR, Gollnick SO, Oseroff AR, Gibson SL, Hilf R and Detty MR. J. Med. Chem. 2000; 43: 2403-2410.

5. Hilmey DG, Abe M, Nelen MI, Stilts CE, Baker GA, Baker SN, Bright FV, Davies SR, Gollnick SO, Oseroff AR, Gibson SL, Hilf R and Detty MR. J. Med. Chem. 2002; 45: 449-461.

6. Abe M, Hilmey DG, Stilts CE, Sukumaran DK and Detty MR. Organometallics 2001; 21: 2986-2992.

7. Abe M, Detty MR, Gerlits OO and Sukumaran DK. Organometallics 2004; 23: 4513 -4518.

8. You Y, Gibson SL, Hilf R, Davies SR, Oseroff AR, Roy I, Ohulchanskyy TY, Bergey EJ and Detty MR. J. Med. Chem. 2003; 46: 3734-3747.

9. You Y, Gibson SL, Hilf R, Ohulchanskyy TY and Detty MR. Bioorg. Med. Chem. 2005; 13: 2235-2251.

10. You Y, Gibson SL and Detty MR. Bioorg. Med. Chem. 2005; 13: 5968-5980.

11. Ulman A, Manassen J, Frolow F and Rabinovich D. J. Am. Chem. Soc. 1979; 101: 7055-7059.

12. Ulman A and Manassen J. J. Chem. Soc., Perkin Trans. 1 1979: 1066-1069.

13. Ulman A, Manassen J, Frolow F and Rabinovich D. Tetrahedron Lett. 1978: 167-170.

14. Ulman A, Manassen J, Frolow F and Rabinovich D. Tetrahedron Lett. 1978: 1885-1886.

15. Ulman A and Manassen J. J. Am. Chem. Soc. 1975; 97: 6540-6544.

16. Lee C-H, Park J-Y and Kim H-J. Bull. Korean Chem. Soc. 2000; 21: 97-100.

17. Heo P-Y, Shin K and Lee C-H. Tetrahedron Lett. 1996; 37: 197-200.

18. Narayanan SJ, Sridevi B, Chandrashekar TK, Vij A and Roy R. J. Am. Chem. Soc. 1999; 121: 9053-9068.

19. Srinivasan A, Mahajan S, Pushpan SK, Ravikumar M and Chandrashekar TK. Tetrahedron Lett. 1998; 39: 1961-1964.

20. Pushpan SK, Narayanan JS, Srinivasan A, Mahajan S, Chandrashekar TK and Roy R. Tetrahedron Lett. 1998; 39: 9249-9252.

21. Punidha S, Agarwal N, Burai R and Ravikanth M. Eur. J. Org. Chem. 2004: 2223-2230.

22. Agarwal N, Hung C-H and Ravikanth M. Eur. J. Org. Chem. 2003: 3730-3734.

23. Kano K, Fukuda K, Wakami H, Nishiyabu R and Pasternack RF. J. Am. Chem. Soc. 2000; 122: 7494-7502.

24. Hunter CA and Sanders JKM. J. Am. Chem. Soc. 1990; 112: 5525-5534.

25. Allen Frank H. Acta Crystallogr., Sect. B: Struct. Sci. 2002; 58: 380-388.

26. Gupta I, Hung CH and Ravikanth M. Eur. J. Org. Chem. 2003: 4392-4400.

27. Latos-Grazynski L, Lisowski J, Szterenberg L, Olmstead MM and Balch AL. J. Org. Chem. 1991; 56: 4043-4045.

28. Agarwal N, Mishra SP, Kumar A, Hung CH and Ravikanth M. Chem. Commun. (Cambridge, U. K.) 2002: 2642-2643.

29. Aitken RA and Garnett AN. J. Chem. Soc., Perkin Trans. 1 2000: 3020-3021.

30. In APEX2 and SAINT-Plus, Area detector control and integration software, Ver. 1.0-27


Y. YOU ET AL.8

Bruker Analytical X ray Systems: Madison, Wisconsin, USA, 2004.

31. In SHELXTL, An integrated system for solving, refining and displaying crystal structures from

diffraction data, Ver. 6.14 Bruker Analytical X-ray Systems: Madison, Wisconsin, USA, 2003.

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www.elsevier.com/locate/jphotobiol

Journal of Photochemistry and Photobiology B: Biology 85 (2006) 155–162

Phototoxicity of a core-modified porphyrin and induction of apoptosis

Youngjae You a,*, Scott L. Gibson b, Michael R. Detty a

a Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY 14260-3000, United Statesb Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 607, Rochester, NY 14642, United States

Received 4 April 2006; received in revised form 20 June 2006; accepted 3 July 2006

Abstract

A core-modified porphyrin, 5-phenyl-10,15-bis(carboxylatomethoxyphenyl)-20-(2-thienyl)-21,23-dithiaporphyrin (IY69) was studiedin vitro for photodynamic activity under a variety of experimental protocols. Variables included the cell line (the rodent mammary tumorcell line R3230AC or the human breast cancer cell line MCF-7), light fluence, time of exposure of the cell cultures to IY69, and the timepost-irradiation for cell counting. The length of time cell cultures were exposed to IY69 impacted cellular accumulation and cellular local-ization, phototoxicity, and the apparent mode of cell death – apoptosis vs. necrosis.� 2006 Elsevier B.V. All rights reserved.

Keywords: Core-modified porphyrin; Breast cancer; Photodynamic therapy; Apoptosis

1. Introduction

Photodynamic therapy (PDT) is a promising strategyfor the treatment of cancer [1–4]. PDT has regulatoryapproval from numerous agencies in the US, Canada,Japan, Great Britain, and Europe for treating a varietyof human malignancies [1–5]. PDT requires three compo-nents: a photosensitizer, oxygen, and light. PDT is mademore effective by increasing the selectivity of the photo-sensitizer for targeted tissues and by the selective deliveryof light via fiber optics. Singlet oxygen is the primaryphototoxic species generated by most photosensitizersupon irradiation and the damage induced by singlet oxy-gen results first in injury to cellular function and struc-ture, and ultimately in cell death and regression oflesions [6]. One major focus of PDT research has beendirected towards synthesizing more effective photosensitiz-ers [7]. The criteria for an effective photosensitizer are thatit (1) is chemically pure and of known composition, (2)has minimal dark toxicity, (3) has preferential uptake

1011-1344/$ - see front matter � 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.jphotobiol.2006.07.002

* Corresponding author. Present address: Department of Chemistry &Biochemistry, South Dakota State University, Brookings, SD 57007,United States. Tel.: +1 605 688 6905; fax: +1 605 688 6364.

E-mail address: [email protected] (Y. You).

Please cite this article as: Youngjae You et al., Phototoxicity of a cPhotochemistry and Photobiology B: Biology (2006), doi:10.1016/j.

and/or retention by tissues of interest, (4) has rapid excre-tion leading to low systemic toxicity, (5) has high quan-tum yield for the generation of singlet oxygen (1O2),and (6) has a strong absorbance with a high extinctioncoefficient in the 600–900 nm range where penetration oflight into tissue is maximal [8].

We have prepared new photosensitizers, the 21,23-core-modified porphyrins, that possess physical and photophys-ical properties that are desirable for PDT [9–11]. Thesemolecules are prepared via flexible synthetic schemes thatallow the photosensitizers to be tailored for specific appli-cations. The substitution of the heavy atoms S or Se forthe core nitrogen atoms of natural porphyrins providesunique physicochemical properties. The core-modified por-phyrins do not bind metals due to the larger atomic sizes ofS or Se in the 21- and 23-positions [9]. More importantly, ared-shift in light absorption from 630 to 690 nm occurs.These compounds have low dark toxicity as evidenced inearlier experiments where skin photosensitivity was mini-mal in a murine model, following exposure to a disulfo-nated core-modified porphyrin and light [9]. Our previousstudies of core-modified porphyrins in vitro demonstratedthat 5-phenyl-10,15-bis(4-carboxylatomethoxyphenyl)-20-(2-thienyl)-21,23-dithiaporphyrin (IY69) showed the great-est potential as a photosensitizer [12].

ore-modified porphyrin and induction of apoptosis, Journal ofjphotobiol.2006.07.002.

mailto:[email protected]

156 Y. You et al. / Journal of Photochemistry and Photobiology B: Biology 85 (2006) 155–162

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Photodynamic responses are highly dynamic processesand experimental factors impact biological results bothin vitro and in vivo. We report our initial biologicalstudies in vitro for PDT with IY69 varying several exper-imental variables: cell line, concentration of the photo-sensitizer, fluence of light, and time. To examine thepathway of cell death, we determined the induction ofapoptosis after PDT with IY69. We find that the photo-toxicity and the extent of apoptotic cell death using IY69

as a photosensitizer are significantly affected by the timefor which cultured cells are incubated with IY69 and theconcentration of IY69.

2. Materials and methods

2.1. Chemicals and reagents

The detailed synthesis of compound IY69 appears inan earlier report [12]. Stock solutions of compoundIY69 were prepared in DMSO at 2 · 10�3 M. Serial dilu-tions were made in sterile doubly distilled water for addi-tion to culture medium. Solvents and reagents were usedas received from Sigma–Aldrich Chemical Co. (St. Louis,MO) unless otherwise noted. The Cell Death DetectionELISAPLUS assay kit for the detection of apoptosis waspurchased from Roche Diagnostics GmbH (Penzberg,Germany). Cell culture medium and antibiotics were pur-chased from GIBCO (Grand Island, NY). Fetal bovineserum (FBS) was obtained from Atlanta Biologicals(Atlanta, GA).

2.2. Cells and culture conditions

The cell lines used for these studies were the R3230ACrat mammary adenocarcinoma cell line and the humanbreast tumor cell line MCF-7. The cells were maintainedin passage culture on 60 mm diameter polystyrene dishes(Becton Dickinson, Franklin Lakes, NJ) in 3.0 mL ofminimum essential medium, a-MEM for the R3230ACcells and Dulbecco’s modified essential medium (D-MEM) for the MCF-7 cells, supplemented with 10%FBS, 50 U/mL penicillin G, 50 lg/mL streptomycin,and 1.0 lg/mL Fungizone (complete medium). Only cellsfrom passages 1 to 10 were used for experiments andcells from passages 1 to 4, stored at �86 �C, were usedto initiate cultures. Cultures were maintained at 37 �Cin a 5% CO2 humidified atmosphere (Forma Scientific,Marietta, OH). Passage was accomplished by removingthe culture medium, adding a 1.0 mL solution containing0.25% trypsin, incubating at 37 �C for 2–5 min to removethe cells from the surface followed by seeding new cul-ture dishes with an appropriate number of cells in3.0 mL of medium. Cell counts were performed using aparticle counter (model ZM, Coulter Electronics, Hia-leah, FL). Cell doubling times were approximately 20 hfor the R3230AC cells and approximately 29 h for theMCF-7 cells.


2.3. Determination of intracellular accumulation

The amount of intracellular dye was determined usingthe fluorescence emission of compound IY69 at 720 nm.Cultured R3230AC or MCF-7 cells were seeded on theinner wells of 96-well plates in the appropriate medium atcell densities ranging from 2 to 3 · 104 cells/well and incu-bated for 24 h. Cells were attached at this point as con-firmed by microscopic evaluation. Compound IY69 wasadded at 5 · 10�6 M and incubated with the cell mono-layer for selected periods from 1 to 24 h. The mediumwas removed, cells were washed twice with 0.9% NaCland 0.2 mL of 25% Scintigeste were added to the mono-layer and incubated for 1 h at 37 �C. The fluorescence inthe cell digests was determined using the multi-well fluores-cence plate reader. Excitation at 440 nm produced a peakemission at 720 nm which was used to determine intracellu-lar concentration of IY69. Cell numbers were determinedas above and the intracellular accumulation of IY69 wascalculated from a fluorescence standard curve generatedfrom known concentrations of IY69 dissolved in Scinti-geste. The cellular concentrations of IY69 are expressedas femtomole/cell.

2.4. Determination of dark- and phototoxicity (MTT assay)

Cytotoxicity (dark- or phototoxicity) was determined byMTT assay [13]. Briefly, cells (1.0–1.2 · 104 cells forR3230AC; 5–7 · 103 cells for MCF-7 cells) in 190 lL com-plete medium were plated on the inner wells of 96 wellplates. Following incubation of cultured R32320AC orMCF-7 cells with various concentrations of IY69 for 24 h,the medium was removed, cells were washed twice with0.2 mL of 0.9% NaCl, and 0.2 mL/well of medium minusFBS and phenol red (clear medium) was added. The plates,with lids removed, were positioned on a orbital shaker(LabLine, Melrose Park, IL) and exposed for various time(less than 1 h) to broadband visible light (350–750 nm)delivered at 1.4 mW cm�2 from a filtered 750 W halogensource defocused to encompass the whole 96 well plate.The culture plates were gently orbited on the shaker in orderto ensure uniform illumination of all of the wells on theplate. The clear medium was then removed, 0.2 mL of freshcomplete medium was added and cultures were incubated at37 �C for various times up to 24 h in the dark. To determinedark-toxicity, cell monolayers were also maintained in thedark undergoing the same medium changes and dye addi-tions as those that were irradiated. Cytotoxicity, eitherdark- or photo-toxicity, was determined using the MTTassay 24 h after the irradiation and expressed as the percentof controls, cells exposed to neither porphyrins nor light.

2.5. Cell counting with a particle counter

To determine cell number with or without irradiation,cells were detached with 1· trypsin–EDTA and an aliquotof 0.2 mL trypsin solution was added to each well. The


ll

0.8

1.0

Y. You et al. / Journal of Photochemistry and Photobiology B: Biology 85 (2006) 155–162 157

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plates were incubated at 37 �C until the cells lifted from thesurface (approximately 5 min). Cell counts were performedusing a particle counter (Model ZM, Coulter Electronics,Hialeah, FL, USA).

Time (h)

0 6 12 18 24

ec/elo

mot

meF

0.0

0.2

0.4

0.6

Fig. 1. Time course of intracellular accumulation of IY69. CulturedR3230AC cells were incubated with 5 · 10�6 M of IY69 for various timesand the intracellular concentration of IY69 was determined from itsfluorescence. Cell culture conditions, fluorescence determination andcalculation of intracellular dye content are detailed in Section 2. Each datapoint represents the intracellular concentration of IY69, expressed asfemtomole/cell, determined in 3 separate experiments performed induplicate, error bars are the SEM.

2.6. Photodynamic treatment and determination of apoptoticcell death and number of cells

Apoptotic cell death was determined by measuring theDNA fragments (nucleosomes) using the Cell Death Detec-tion ELISAPLUS kit and the details followed the instruc-tional manual (Roche Diagnostics, Cat. No. 11 920 685001) [14]. Cell numbers were determined with the Coultercounter as described above. R3230AC cells were platedon the inner wells of 96 multi-well tissue culture plates ata concentration of 5 · 103 cells per well in 190 lL completemedium and incubated for 24 h. Various concentrations ofIY69, 5 · 10�8 –4 · 10�6 M, were added to the monolayersand incubated for 4 or 24 h at 37 �C in the dark. Followingthe incubation periods the medium containing IY69 wasremoved, 200 lL of clear medium was added to the wellsand cells were irradiated as described in the section formeasurement of phototoxicity. Clear medium was replacedwith 200 lL complete medium, the cells were incubatedadditionally for 2 h. Cells were counted with a particlecounter to determine the number of live cells. To determineapoptotic cell death, cells were lysed and placed into thewells of a streptavidin-coated 96 well plate. By adding amixture of anti-histone–biotin and anti-DNA–peroxidase,mono- and oligonucleosomes were captured forming sand-wich immunocomplexes. Absorbances at 405 and 490 nmwere measured after adding ABTS [2,2 0-azinodi-(3-ethyl-benzthiazoline sulfonate)] substrate. The difference ofabsorbances (ABS) per 104 cells was calculated for the unitof nucleosomal production

Unit of nucleosomal production

¼ ABS½405–490 nm�=104 cells

2.7. Statistical analysis

Statistical analyses were performed using the Student’st-test. For all tests, P-value of less than 0.05 was consideredto be a statistically significant difference.

3. Results and discussion

3.1. Time dependent intracellular accumulation into cultured

R3230AC or MCF-7 cells

Cultured R3230AC cells were exposed to 5 · 10�6 MIY69 for various times. Such a high concentration wasemployed due to the low intrinsic fluorescence of IY69

(/F = 0.009) [12]. The intracellular concentration of IY69

reached a plateau after 6–7 h (Fig. 1). Longer incubationtimes gave no significant increase in cellular accumulation.


3.2. Dark- and phototoxicity towards cultured R3230AC or

MCF-7 cells

No dark toxicity was observed upon incubation of eitherR3230AC or MCF-7 cells with up to 1 · 10�6 M IY69 for24-h as shown in Fig. 2. However, both cell lines showedsignificant phototoxicity upon irradiation following expo-sure to IY69 at concentrations between 1 · 10�7 M and1 · 10�6 M. Comparable concentrations of IY69 were morephototoxic toward R3230AC cells than toward MCF-7cells, which is consistent with the lower intracellular accu-mulation of IY69 in the MCF-7 cells (data not shown).However, we cannot exclude other possibilities includinga lower level of caspase-3 in MCF-7 cells [15] since at lowerconcentrations of IY69 (<3 · 10�7 M), apoptosis appearsto be an important mechanism of cell death as describedbelow in Section 3.6. It has been demonstrated thatMCF-7c3 cells, which express more caspase-3 than MCF-7v cells, are more sensitive to apoptotic cell death withthe photosensitizer Pc4 than MCF-7v cells although theoverall efficacy either in vitro or in vivo is not related tocaspase-3 levels in MCF-7 cells [16,17].

3.3. Porphyrin and light dose dependency of phototoxicity in

cultured R3230AC cells

The data displayed in Fig. 3 demonstrate that phototox-icity towards cultured R3230AC was both porphyrin andlight-dose dependent. It appears that the minimum dosesrequired to achieve 40% phototoxicity are 1 · 10�7 MIY69 combined with at least 3.75 J/cm2 of broad bandwhite light delivered at 1.4 mW/ cm2. Although IY69 doesnot absorb the whole range of broad-band light, its absorp-tion is expected to be proportional to the total fluence of


Conc. (μM)

0.01 0.1 1

lavivru

S %

0

20

40

60

80

100

120

R3230AC dark-toxicityMCF-7 dark-toxicityR3230AC phototoxicityMCF-7 phototoxicity

1) plate cells 2) add IY69 3) irradiate0 h 24 h 48 h 72 h

4) MTT

Fig. 2. Dark- and phototoxicity of compound IY69 towards cultured R3230AC cells (closed symbols) and MCF-7 cells (open symbols). Cell culture andirradiation conditions are detailed in Section 2. Each data point represents the mean of at least 3 separate experiments performed in duplicate, bars are theSEM. Data are expressed as the percent of viable cells compared to control cells (cells not exposed to compound IY69 or light).

0

20

40

60

80

100

120

0.1 0.2 0.4Conc (μM)

lavivru

S %

1.25 J/cm2

2.50 J/cm2

5.00 J/cm2


4) MTT

Fig. 3. Porphyrin and light dose related phototoxicity towards cultured R3230AC cells. Cell culture, irradiation conditions and viability determinationsusing MTT are detailed in Section 2. Each data point represents the mean of at least 3 separate experiments performed in duplicate, bars are the SEM.Data are expressed as the percent of toxicity compared to control cells (cells not exposed to compound IY69 or light).


ARTICLE IN PRESS

the light. The data also show that a relationship betweenporphyrin dose and total fluence exists. For example,increasing the porphyrin dose allows for a concomitantdecrease in the total amount of light delivered to achievethe same level of phototoxicity.

3.4. Effects of time after irradiation on phototoxicity towards

cultured R3230AC cells

The data displayed in Fig. 4 represent the phototoxicitytoward R3230AC cells at various times after the irradiationwith IY69. Cell counts were determined at different times,1, 6, 12, and 24 h after irradiation and data are reported


as the ratio of the number of live cells in treated samplesto the number of live cells in controls. The data demon-strate that the cell number was significantly decreased by6 h after irradiation, compared to the number of controlcells, for compound IY69 at 1 · 10�7 M or greater concen-tration. IY69 concentrations higher than 2 · 10�7 M pluslight continued to elicit a significantly greater decrease incell viability over the whole 24 h period. Concentrationsless than 5 · 10�8 M IY69 imposed little or no significantphototoxicity until 24 h after irradiation.

Interestingly enough, at 1 · 10�7 M IY69 cell numberwas constant up to 12 h after irradiation. To keep the cellnumber constant up to 12 h, there were two possibilities.


0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 6 12 18 24Time after irradiation (h)

sllec laitini f

o .o

n / sllec evil fo .

oN

0.0 2.5x10-81.0x10-7 2.0x10-74.0x10-7


4) count

Fig. 4. Time course of R3230AC phototoxicity after irradiation of monolayers exposed to compound IY69. Cell culture and irradiation conditions aredetailed in Section 2. Each data point represents the mean of at least 2 separate experiments performed in triplicate, bars are the SEM. Data are expressedas ratios of cell numbers counted at various times after irradiation with compound IY69 at concentrations ranging from 0 to 4 · 10�7 M.


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First, the initial cells did not proliferate without cell death,i.e. cytostatic effect. Secondly, some cells died but the othercells doubled quickly to compensate the number of deadcells. Gibson et al. reported cytostatic effect of photody-namic therapy of 0.05 mM d-ALA with 30 mJ/cm�2 irradi-ation [18]. It is not clear which is the case in thisexperiment. However, cell number slightly increased at24 h although overall phototoxicity at 24 h was 54% com-pared to the control, which means cells regain their prolif-erate capacity. It will be interesting to observe the longterm effects on phototoxicity and more detailed mechanis-tic study which will elucidate dynamic cellular responses tolow dose photodynamic treatment with IY69.

1) plate cells 2) add IY690 h 24 h

0

20

40

60

80

100

120

0 6 12

lavivru

S %

Time of incubation with IY69 b

Fig. 5. Effect of time of exposure of cultured R3230AC cells to compound IY6

and cytotoxicity determinations using MTT are detailed in Section 2. Each dataduplicate, bars are the SEM. Data are expressed as the percent of control cell


3.5. Effect of incubation time of cells with IY69 before

irradiation on phototoxicity

Phototoxicity was determined for cultured R3230ACcells incubated with compound IY69 for various timesprior to irradiation. The cells were exposed to IY69 for 1,3, 5, 7, 9, 12, 18, or 24 h, then, irradiated with 5 J/cm�2

of broad-band light. Phototoxicity was determined 24 hafter the irradiation. The data displayed in Fig. 5 show thatthe longer cells are incubated with compound IY69, themore effective the subsequent light treatment becomes. Athigher concentrations, e.g. (1 · 10�6 M), significant levelsof cytotoxicity can be reached at much shorter incubation

3) irradiate48 h 72 h

4) MTT

1.0x10-72.0x10-71.0x10-6

18 24

efore irradiation (h)

9 prior to irradiation on phototoxicity. Cell culture, irradiation conditionspoint represents the mean of at least 3 separate experiments performed incytotoxicity (cells not exposed to compound IY69 or light).



ARTICLE IN PRESS

intervals, within 6 h with incubation time-dependent man-ner. If we assume that the kinetics of uptake at these con-centrations, 1, 2, or 10 · 107 M, is similar to that at5 · 10�6 M (Fig. 1), the time-dependent phototoxicity ofIY69 up to 6 h is consistent with the time-dependent con-centration of IY69 in cells.

Incubation time-dependency of phototoxicity was alsoabsorbed after 6 h in cells treated with 2 or 1 · 10�7 MIY69. The difference of phototoxicity at 6 and 24 h with1 · 10�7 and 2 · 10�7 M cannot be explained by the differ-ence in concentration because the amounts of IY69 at 6and 24 h are expected to be similar. In our fluorescencemicroscopic study, the distributions of fluorescence fromIY69 in cells at 4 and 24 h after addition were different:most of florescence seemed evenly distributed throughoutcells after 4 h (data not shown). On the other hand, local-ized bright spots of the fluorescence were absorbed in cellsat 24-h after the addition of IY69 without staining nuclei[12]. The sites of localization of photosensitizers are thesites of initial photodamage by singlet oxygen generated

a

0

0.5

1

1.5

2

2.5

0 5 10 15 20

Conc ( x10

01x1 /]094-504[ S

BA

4sllec

b

0

20

40

60

80

100

120

140

0 5 10 15 20

Conc ( x10

lavivru

S %

1) plate cells 2) add IY690 h 24 h

Fig. 6. Determination of cell number and apoptosis. Cell culture and irradiatioof at least 3 separate experiments performed in duplicate, bars are the SEM.


by photodynamic therapy: it has been generally believedthat life time of singlet oxygen is short, 0.03–0.18 lsin vivo, thus diffusion distance of singlet oxygen is very lim-ited [19]. Thus, we presume that the localization of IY69 inspecific sites inside cells with longer incubation make thephotosensitizer more efficient in expressing phototoxicity.

3.6. Effect of incubation time of cells with IY69 on induction

of apoptotic cell death

We determined the degree of photo-induced apoptosisafter 4- or 24-h incubation of cultured R3230AC cells withvarious concentrations of IY69 as well as the number ofcells in parallel for phototoxicity. Apoptotic cell deathwas measured using the Cell Death Detection ELISAPLUS

kit provided by Roche diagnostics. This kit determinesthe presence of mono- and oligonucleosomes in the cytosolof cell lysates as one representative indication of the DNAdegradation following the induction of apoptosis [20,21].The data displayed in Fig. 6 reveal the importance of both

25 30 35 40

-7 M)

24 h

4 h

25 30 35 40

-7 M)

24 h4 h

3) irradiate48 h 51 h

4) count/detect nucleosomes

n conditions are detailed in Section 2. Each data point represents the mean



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the concentration of IY69 and the time of exposure of cul-tured R3230AC cells to IY69 on both phototoxicity andinduction of apoptosis.

The level of phototoxicity was dependent on the concen-tration of IY69 for cells exposed for either 4 h or for 24 h tothe photosensitizer (Fig. 6a). For any given concentration,24-h exposure to the photosensitizer gave increased photo-toxicity relative to a 4-h exposure. As an example, exposureto 5 · 10�7 M IY69 and light gave nearly 100% phototox-icity while a 4-h exposure gave around 40% phototoxicity.

The production of mono- and oligonucleosomes due tothe induction of apoptosis was affected by the concentrationof IY69 (Fig. 6b). However, the effect did not solely dependon the concentration of IY69. There were optimal concen-trations for the maximal production of nucleosomes forboth 4 and 24-h exposure conditions, 0.3 and 2.1 units(ABS[405–490]/1 · 104 cells) at 1 · 10�6 and 2 · 10�7 MIY69, respectively. At higher concentrations above the opti-mal conditions, the production of nucleosomes decreased(Fig. 6b). The production of nucleosomes reached minimalpoints, 0.53 and 0.094 units at high concentrations, 5 · 10�7

and 4 · 10�6 M IY69 for 4 and 24-exposures. The concen-tration of photosensitizers in cells was reported as one ofthe main factors influencing pathway of cell death afterPDT [22–26]. It was consistent in PDT with IY69 thatthe mode of cell death shifted pathway from pro-apoptoticto pro-necrotic as concentration of a photosensitizerincreased.

The other interesting result was the effect of exposuretime of IY69 to the cells on induction of apoptosis. Overall,PDT with 24-h exposure of IY69 produced more nucleo-somes than that with 4-h exposure. At the optimal condi-tions, longer exposure of IY69 to R3230AC cells at lowerconcentration, 2 · 10�7 M for 24 h, produced the morenucleosomes than shorter exposure at higher concentra-tion, 1 · 10�6 M for 4 h. The production of nucleosomesat 2 · 10�7 M with 24-h exposure was seven times theamount of nucleosomes at 1 · 10�6 M with 4-h exposure:2.1 vs. 0.3 units. Considering this result with the intracellu-lar localization 24 h after addition of IY69 [12], it is likelythat the sites of localization of IY69 with 24-h exposurewere important organelles for apoptosis. This hypothesisis supported by previous reports where the sites of localiza-tion of photosensitizers in cells were primary photodamagesites, consequently important in determining cell deathpathway in PDT: apoptosis or necrosis [6,22,27–31].

4. Conclusion

The new photosensitizer, IY69, was phototoxic towardsboth cultured R3230AC rat mammary tumor cells andMCF-7 human breast cancer cells. The phototoxicitytoward R3230AC cells exposed to IY69 and light wasdependent on various experimental conditions such as con-centration of the IY69, total fluence of light, the length oftime post irradiation, and exposure time of IY69 to cul-tured R3230AC cells. Especially, the time of exposure of


IY69 to R3230AC cells was a main factor for cellular con-centration, localization, and cell death mechanism afterirradiation. We are thus under process to find molecularmechanistic and pharmacokinetic profiles of photodamageand localization in PDT with IY69.

Acknowledgement

This research was supported by the Department of De-fense [Breast Cancer Research Program] under award num-ber W81XWH-04-1-0500. Views and opinions of, andendorsements by the author(s) do not reflect those of theUS Army or the Department of Defense.

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